In this project we propose to study the subcellular distribution of the adenylate cyclase from the human astrocytoma cell line #132-1N1, and from the normal (WI-38) and transformed (VA-13 and Va-2) human lung fibroblast cell line and compare the stimulation of the adenyl cyclase in the whole lysates and various subcellular fractions with the stimulation of cAMP accumulation in the intact cells. Continued attempts will be made to establish the critical aspects of adenosine and adenine nucleotide structure which confer specificity for the receptor in these intact cultured cell lines and in the broken cell preparations of adenylate cyclase from these cells. Adenosine stimulation is inhibited by methylanthines and attempts will be made to determine the structural specificity of inhibitors of the adenosine stimulation. The adenosine stimulation of adenylate cyclase is very labile and attempts will be made to establish the optimum conditions for preservation of the adenosine receptor during cell lysis and preparation of subcellular fractions. We will carry on the screening of various cultured cell lines for the effects of adenosine on cAMP metabolism. Using the chinese hamster ovary cell line (CHO),a comparison will be made of the response of the cAMP system to adeosine with varied cell culture conditions; eg. monolayer growth versus cell suspension culture. We will continue to investigate the importance of adenosine and adenine nucleotide regulation of cAMP metabolism in guinea pig and rat heart muscle, and attempt to develop preparations of vascular smooth muscle and guinea pig taenia coli suitable for evaluating the effect of adenosine. BIBLIOGRAPHIC REFERENCES: McGuire, R. F., and Clark, R. B., Isolation of Two Populations of Plasma Membranes from Cultured Human Glioma Cells with Epinephrine and Adenosine Sensitive Adenylate Cyclase, Fed. Proc. Abs. #1368, (1976). Clark, R. B. and Seney, M. Natlie, J. Biol. Chem. June (1976) Regulation of Adenylate Cyclase from Cultured Human Cell Lines by Adenosine.